Hello all,
I am relatively new to small RNA seq (have only worked with regular RNA seq), and I had a question about my workflow. I am looking for differential expression of miRNAs between 2 sample types - not interested at the moment in novel miRNA discovery. I have 50 bp paired-end reads from an Illumina HiSeq using a library prepared by the Illumina Truseq Small RNA protocol.
I was planning to use cutadapt to trim the adapters from the forward and reverse reads since cutadapt has the ability to work with paired end reads. My question comes in the maximum and minimum sizes of the reads to keep. It is my understanding that mature miRNAs range in sizes around 17 to 25 nt long, so would it be correct to have cutadapt only keep reads with a minimum of 15 nt and a maximum of 30 nt long after adapter trimming in order to help eliminate other RNAs present in the data? I was planning to use bowtie to map the data to miRBase after the trimming.
Another alternative I was thinking of was only keeping those reads in which the forward and reverse read are perfectly complementary to each other after trimming, but I was unsure of this method and/or how to perform it.
Thanks in advance!
I am relatively new to small RNA seq (have only worked with regular RNA seq), and I had a question about my workflow. I am looking for differential expression of miRNAs between 2 sample types - not interested at the moment in novel miRNA discovery. I have 50 bp paired-end reads from an Illumina HiSeq using a library prepared by the Illumina Truseq Small RNA protocol.
I was planning to use cutadapt to trim the adapters from the forward and reverse reads since cutadapt has the ability to work with paired end reads. My question comes in the maximum and minimum sizes of the reads to keep. It is my understanding that mature miRNAs range in sizes around 17 to 25 nt long, so would it be correct to have cutadapt only keep reads with a minimum of 15 nt and a maximum of 30 nt long after adapter trimming in order to help eliminate other RNAs present in the data? I was planning to use bowtie to map the data to miRBase after the trimming.
Another alternative I was thinking of was only keeping those reads in which the forward and reverse read are perfectly complementary to each other after trimming, but I was unsure of this method and/or how to perform it.
Thanks in advance!
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